Given the well-known pathogenicity of Staphylococcus aureus, the emergence of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) is causing increased public health concerns. More than 60% of S. aureus-associated hospital acquired infections are caused by MRSA, which has increasingly spread beyond healthcare facilities and has emerged as a community pathogen. The annual cost to treat MRSA in hospitalized patients in the U.S. is estimated to be between $3.2 billion to 4.2 billion. The threat of VRSA has been the topic of intensive research since there is a limited drug of choice for MRSA infections. Therefore, there is an urgent need to develop more efficient preventive and/or therapeutic agents to fight this pathogen. We have demonstrated that a staphylococcal putative glycoprotease (Gcp) is a unique antibacterial target since it is cell-wall associated and secreted, essential for survival for both Gram-positive and Gram-negative pathogens. However, the biological function of the putative glycoprotease is still unclear. Without a biochemical readout phenotype, it is difficult for us to develop a high-throughput screen of inhibitors against this novel target. Combined microarray and proteomic approaches with a regulated RNA interference technology, we found that the down-regulation of gcp expression significantly increases the productions of acetolactate synthase, dihydroxy-acid dehydratase and threonine dehydratase, which are located in the same metabolic pathway in S. aureus. Therefore, we hypothesize that these enzymes are the surrogate markers of Gcp and monitoring Gcp's biomarkers will provide a powerful approach to high-throughput screen inhibitors against Gcp. The objective of this proposal is to develop a whole cell-based high throughput assay using these elevated proteins as Gcp's surrogate biomarkers. In specific aim 1, we will construct bioluminescent reporter systems using Gcp's surrogate markers in S. aureus. In specific aim 2, we will perform high-throughput screening of novel chemical leads against this essential putative glycoprotease using the bioluminescent reporter systems. In specific aim 3, we will validate lead compounds to determine their mode of action, specificity, and selectivity. Successful completion of these studies will identify potential inhibitors against this novel, unique antibacterial target, Gcp, as well as provide a novel strategy for high-throughput screening of compounds against other functional unknown targets. Therefore, the proposed studies will provide a novel strategy for high-throughput screening of compounds against novel drug target that will be particularly important to therapeutic interventions for infections and medical practice to fight the emerging antibiotic-resistant S. aureus. PUBLIC HEALTH RELEVANCE: S. aureus is an important community and hospital acquired pathogen causing a wide variety of infections, ranging from superficial skin to life-threatening systemic infections, including pneumonia, endocarditis, septic arthritis and toxic shock syndrome. The recent emergence of multiple antibiotic- resistant strains of S. aureus, especially methicillin-resistant S. aureus and vancomycin-intermediate resistant S. aureus, is causing great concern in the public health community. There is an urgent need for new classes of antibiotics and potent vaccines to fight infections caused by S. aureus. The outcome of our proposed project will identify potential lead compounds against a unique, secreted antibacterial target, as well as provide a novel strategy for high-throughput screening of inhibitors against functional unknown targets.